Quantification of HPLC-separated peptides and proteins by spectrofluorimetric detection of native fluorescence and mass spectrometry

Due to relatively low reproducibility of the ionization and differences when using buffers as mobile phases, the quantitative analysis by electrospray ionization mass spectrometry (ESI-MS) can be often challenging. In the present study, the native fluorescence of phenylalanine, tyrosine, and tryptophan was investigated as an improvement tool for the analytical quantification of peptides and proteins by HPLC–ESI-MS. Natively fluorescent amino acids as well as peptides, proteins, and protein digests were successfully separated by HPLC, and quantified with a spectrofluorimetric detector and ESI-MS. The two detectors were connected in series and enabled the sequential measurements of the fluorescence intensities as well as the measurements of the ion signals and mass spectral characterization of separated polypeptides. Fluorescence detector provided better linearity and repeatability of quantification than mass spectrometer, and similar limits of detection for most of biomolecules analyzed. The fluorescence signal was linear over 3–4 orders of magnitude with limits of detection in picomole or high femtomole range, depending on nature and number of natively fluorescent amino acid residues present in the analyzed polypeptides. Hence, native fluorescence of phenylalanine, tyrosine, and tryptophan can be used as a label-free methodology to facilitate quantification of peptides and proteins by LC–ESI-MS.

► Sequential native fluorescence–ESI-MS quantification of HPLC-separated polypeptides is enabled. ► Peptides and proteins containing all natively fluorescent amino acids are quantified. ► Figures of merit for spectrofluorimetric and ESI-MS quantifications of natively fluorescent biomolecules are complementary. ► Native fluorescence provides better linearity and repeatability of quantification than ESI-MS. ► This is a label-free technique, which can facilitate quantification of peptides and proteins by LC–ESI-MS.